STEM CELL CULTURE

STEM CELL CULTURE

1. STEM CELL CULTURE Done by : Sumaiah Alghamdi Hend AL-subaie Ala'a Al-Ruwaisan Norah Alhoshani Submitted to :Dr. zaineb Qorni 1435-1436

2. Objective STEM CELL HISTORY Stem Cell Definitions Why are stem cells important Classification of stem cells based on their dividing capacity Culturing Stem Cells Embryonic Bone marrow Umbilical Human Cord culture References

3. STEM CELL HISTORY W e are currently facing an unprecedented level of public interest in research on embryonic stem cells, an area of biomedical research that until recently was small, highly specialized and of limited interest to anyone but experts in the field. Real and imagined possibilities for the treatment of degenerative and other diseases are of special interest to our rapidly ageing population; real and imagined associations of stem cells to cloning, embryos and reproduction stir deeply held beliefs and prejudices. The conjunction of these factors could explain the recent sudden interest in embryonic stem cells but we ought to remember that this research has a long and convoluted history, and that the findings described today in the scientific and popular press are firmly grounded in research that has been going on for several decades. Here I briefly recapitulate this fascinating history.

4. STEM CELL HISTORY 1981 University San Francisco and Cambridge. 1995   University of Wisconsin isolate. 1998 Dr . James A. Thompson Dr. John Gearhart 2001 Massachusetts scientists . 2005 Harvard University 2007 University and Harvard University 2012 University of Queensland 2013 Human Liver Created from Stem Cells. 2014 British scientists make custom-made body parts using stem cells

5. Stem Cell Definitions O ne of the human body's master cells, with the ability to grow into any one of the body's more than 200 cell types. Stem cells are unspecialized (undifferentiated) cells that are characteristically of the same family type (lineage). They retain the ability to divide throughout life and give rise to cells that can become highly specialized and take the place of cells that die or are lost. Stem cells contribute to the body's ability to renew and repair its tissues. Unlike mature cells, which are permanently committed to their fate, stem cells can both renew themselves and create new cells of whatever tissue they belong to (and other tissues). Bone marrow stem cells, for example, are the most primitive cells in the marrow. From them all the various types of blood cells are descended. Bone marrow stem cell transfusions (or transplants) were originally given to replace various types of blood cells.

6. Why are stem cells important S tem cells represent an exciting area in medicine because of their potential to regenerate and repair damaged tissue. Some current therapies, such as bone marrow transplantation, already make use of stem cells and their potential for regeneration of damaged tissues. Other therapies are under investigation that involves transplanting stem cells into a damaged body part and directing them to grow and differentiate into healthy tissue.

7. Classification of stem cells based on their dividing capacity PLURIPOTENT STEM CELLS .(FIGURE1) TOTIPOTENT STEM CELLS MULTIPOTENT STEM CELLS .(FIGURE)2 ( F igure 1) ( F igure 2)

8. What are the major types of stem cells?! Embryonic stem cells (ES cells). Adult stem cells. Induced Pluripotent Stem Cells (iPSCs)

9. There are three general classifications of stem cells: adult stem cells (ASCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) ( F igure 2) .

10. Embryonic stem cells (ES cells). cells to zygote to blastocyst to preg - nancy, birth and adult.

11. Adult Stem Cells Hematopoietic stem cells(HSCs) Neural Stem Cells(NSCs) Mesenchymal Stem Cells(MSCs)

12. Cancer with stem cells Figure8

13. Stem cells with Parkinson's Disease Figure5

14. Induced Pluripotent Stem Cells(IPSs) Figure 9 Figure 10

15. Culturing Stem Cells Embryonic

16. Embryonic Stem Cells (ESC) Embryonic stem cells, as their name suggests, are derived from embryos Are getting stem cells from the inner cell mass ( blastocyle ) Which is touted as the whole-cell efficiency ( totipotent ) , while the outer layer of fabric or food blastocyle be responsible for formation of the placenta and other supporting tissues needed by the fetus Twelve process formation in the womb While the internal cells creates various body tissues, including the organism Stem cells are the foundation for every organ , tissue, and cell in the human body.

17. Embryonic Stem Cells

18. D ifferentiation pathways of adult stem cells Normal differentiation pathways of adult stem cells. In a living animal, adult stem cells are available to divide , when needed, and can give rise to mature cell types that have characteristic shapes and specialized structures and functions of a particular tissue. The following are examples of differentiation pathways of adult stem cells that have been demonstrated in vitro or in vivo .

19. Differentiation pathways of adult stem cells Hematopoietic stem cells Mesenchymal stem cells Neural stem cells Skin stem cells Epithelial stem cells

20. Differentiation pathways of adult stem cells

21. Potential Uses for Stem Cells

22. Culturing Stem Cells Embryonic Origin blastocyst of embryo strengths pluripotent (3 germ layers ) self- renewal and high replicative capacity weaknesses immunologic al concerns subject to ethical debate potential for teratoma and teratocarcinoma currently no clinical trial data

23. How to grow embryonic stem cells in vitro Dissect out the uterine horns 1-briefly rinse in 70 ethanol 2- place into a falcon with PBS 3-separate each embryo from placenta and embryonic sac Dissect head and red organsh 1- wash in PBS 2- place all embryos in a clean petri dish

24. How to grow embryonic stem cells in vitro Finely mince the tissue using a sterile razor blade until it becomes .... pipettable 1- add trypsin and dnasel 2- incubate for 15minutes at 37c 3- pipet up and down after each 5 minutes incubation 4- inactivate - trypsin by adding MEF medium 5- centrifuge at 300xg . 5minutes 6- remove the supernatant 7- resuspend cell pellet 8-Freeze the cells after implant

25. Cell differentiation during culture

26. The lack of differentiation of cells during the culture

27. At several stages of generating embryonic stem cell lines operations of cells , scientists tested to see whether these cells show the basic characteristics that make embryonic stem cells . This process is called knowledge Characterization characteristics The laboratories that develop human stem cells use several tests lines . These tests include: Laboratory tests used to identify embryonic stem cells

28. Laboratory tests used to identify embryonic stem cells Planting and replanting of stem cells for several months. Using special techniques to determine the presence of superficial signs of surface markers and found only on the surfaces of undifferentiated cells . One other important tests is the presence of a protein Oct-4 that is formed cells is usually distinguished Microscopically examined chromosomes .   Determine whether it was possible to replant cells after freezing , dissolved and repeat cultivated .

29. Bone marrow Bone marrow is the soft, spongy substance that fills the inner cavities of bones. It is where blood is produced. Tiny spaces in the bone marrow hold blood and stem cells, the primitive cells that are able to grow into various types of blood cells. During a bone marrow harvest, a small part bone marrow is collected, or harvested, to use for transplantation.

30. Bone marrow

31.   Stem Cell Isolation from Murine Bone Marrow Cells Steps: 1-kill mice by CO2 asphyxiation. It is preferable to work with 2-3 mice at one time utilized of 4-5 mice, of which are between 6-10 weeks of age.

32.   Stem Cell Isolation from Murine Bone Marrow Cells Steps : 2- cut limbs from one mouse. Remove skin and as much muscle and connective tissue as possible Cut the limb above the hip and below the ankle joint (including some of the foot); it is important to maintain the bone ends where bone marrow exist, carefully break apart the knee joint and strip remaining connective tissue from both ends .

33.   Stem Cell Isolation from Murine Bone Marrow Cells

34. Stem Cell Isolation from Murine Bone Marrow Cell Steps : 3- wipe bones with your finger after dipped in 70% Ethnol and place bones in dish of sterile PBS on ice. Collect all bones in pool in same dish.  

35. Stem Cell Isolation from Murine Bone Marrow Cell

36. Stem Cell Isolation from Murine Bone Marrow Cell Steps : 4. Move bones into sterile tissue culture hood. Wash bones by sterile PBS 6 times

37. Stem Cell Isolation from Murine Bone Marrow Cell Steps : 5-cut off ends of each bone with scissors (keeping as close to end as possible to extract more bone marrow) and gently place in sterile PBS.

38. Stem Cell Isolation from Murine Bone Marrow Cell Steps : 6-Fill 10cc syringe with prewarmed Complete conditioned media and attach 25 gauge needle With forceps grab one bone. Use syringe to force media through bone shaft to extract all red marrow into plate. Can flip bone over and do from opposite side, Repeat a few times to ensure all marrow is removed. Bone should look very white when finished.

39. Stem Cell Isolation from Murine Bone Marrow Cell

40. Stem Cell Isolation from Murine Bone Marrow Cell Steps : 7- Pipette cell mixture up and down a few time so dissociate cells- can also use syringe to pull large marrow pieces through needle to dissociate further then, Pass cell suspension through cell strainer (70µm size) to remove any large cell clumps or bone particles .

41. Stem Cell Isolation from Murine Bone Marrow Cell Steps 8- Centrifuge the cell suspension at 250 rpm × 8 min, after that , Remove the supernatant and resuspend cell pellet in 20 ml of HBSS

42. Stem Cell Isolation from Murine Bone Marrow Cell Steps: 9-Add Growth medium to a petri dish addthe cell suspension into it .

43. Stem Cell Isolation from Murine Bone Marrow Cell Steps: 10-Incubate cells until cells adhere and look nearly confluent—usually around day 4 of culture for a pool from 4 mice then Remove media and wash once with PBS to remove non adherent cell.

44. Stem Cell Isolation from Murine Bone Marrow Cell Steps: 11-Expand cells until 70%-90% confluent, changing media every 3-4 days . At this point, can either split further for experiments of freeze in liquid nitrogen for further use .

45. Stem Cell Isolation from Murine Bone Marrow Cell Steps: 12-At this time, cells can be plated for different assays including differentiation antibody.

46. Advantages and disadvantages Advantages Clinical safety . efficacy data . Typically lineage  committed  Disadvantages : Limited number  Limited replicative capacity  Lineage  restricted

47. Umbilical Human Cord culture

48. Umbilical Human Cord _ Human umbilical cord is a reach source mesenchymal stem cells (MSCs). _ It is useful and harmless.

49. Umbilical Human Cord _ The extraction techniques that used for umbilical cord: _ Umbilical vein. _ Arteries. _ Wharton’s jelly. _ Whole umbilical cord- UC Non- invasive source.

50. Materials and Methods 1_ isolation of human umbilical cord MSCs: Cut about 28 CM long of umbilical cords from donor. The maternal age about 34, and gestation age from 38_40 weeks. Make gentle massage for harvesting cells from UC vein.

51. The collection process

52. Human Umbilical Cord

53. Materials and Methods Wash the samples hypochlorite solution ( 1:3). Rinsed the samples by PBS (phosphate- buffered saline) . Stored in DMEM low Glucose ( Dulbecco’s Modified Eagle’s Medium) supplemented with 10% fetal bovine serum ( Gibco) and antibiotics ( Penicillin/streptomycin and gentamcin). After those treatment collecting steps transport to laboratory to complete process.

54. Material and methods Wash the umbilical cord blood by PBS three time to remove clots. Add 0.1% of collagenase 3 ml for enzymatic digestion vein and arteries. incubation for 20 minutes. Transfer the content into centrifuge tube. centrifugation tubes at 4000 rpm for 10 min at room temperature .

55. Material and methods Discard the supernatant, and resuspend the pellet. Transfer the content into two 25cm flask, and culture in DMEM, supplement with 10% FBS, 1% antibiotics in 5% CO2 in 37ºC incubator. After 24 hour of incubation nonadherant cell must be removed. The stem cells now has been cultured. 2

56. Centrifugation steps

57. cultured umbilical cord stem cell

58. Advantages of umbilical cord blood 1 _ It is the easiest way for stem cells culture. 2_ There is no risk for donors either child or mother. 3_ Huge number of healthy donors. 4_We can storage the umbilical stem cell for long time. 5_ It has more Histocompatibility between umbilical cord cells comparing with marrowy bone extracted cells. 6_ Required less money.

59. D isadvantages of umbilical cord blood 1_ It could be developing genetic disease which allow to transport the disease to patient. 2_Difficulty for maintain enough stem cells in collected of umbilical cord units. 3_Using more than one sample for only one patient.

60. References http://lebawab.wordpress.com / Xu, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nature 19:971 (2001 ). http:// www.cirm.ca.gov/our-progress/stem-cell-definitions Resource guide for stem cell transplant – Including bone marrow, peripheral blood, and cord blood. www.nbmtlink.org  Accessed 2/2/2012 http:// research.jax.org/faculty/mills/protocols/mesenchymal_stem_cell_isolation.pdf   http:// www.jbmethods.org/jbm/article/view/12/6 http://www.lume.ufrgs.br/bitstream/handle/10183/26823/000758572.pdf